Cystic fibrosis (CF) is a recessive genetic disease that affects approximately 30,000 children and adults in the United States and approximately 30,000 children and adults in Europe. Despite progress in the treatment of CF, there is no cure.
CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes an epithelial chloride ion channel responsible for aiding in the regulation of salt and water absorption and secretion in various tissues. Small molecule drugs known as potentiators that increase the probability of CFTR channel opening represent one potential therapeutic strategy to treat CF. Potentiators of this type are disclosed in WO 2006/002421, which is herein incorporated by reference in its entirety. Another potential therapeutic strategy involves small molecule drugs known as CF correctors that increase the number and function of CFTR channels. Correctors of this type are disclosed in WO 2005/075435, which is herein incorporated by reference in its entirety.
Specifically, CFTR is a cAMP/ATP-mediated anion channel that is expressed in a variety of cell types, including absorptive and secretory epithelial cells, where it regulates anion flux across the membrane, as well as the activity of other ion channels and proteins. In epithelial cells, normal functioning of CFTR is critical for the maintenance of electrolyte transport throughout the body, including respiratory and digestive tissue. CFTR is composed of approximately 1480 amino acids that encode a protein made up of a tandem repeat of transmembrane domains, each containing six transmembrane helices and a nucleotide binding domain. The two transmembrane domains are linked by a large, polar, regulatory (R)-domain with multiple phosphorylation sites that regulate channel activity and cellular trafficking.
The gene encoding CFTR has been identified and sequenced (See Gregory, R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature 347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). A defect in this gene causes mutations in CFTR resulting in cystic fibrosis (“CF”), the most common fatal genetic disease in humans. Cystic fibrosis affects approximately one in every 2,500 infants in the United States. Within the general United States population, up to 10 million people carry a single copy of the defective gene without apparent ill effects. In contrast, individuals with two copies of the CF associated gene suffer from the debilitating and fatal effects of CF, including chronic lung disease.
In patients with CF, mutations in CFTR endogenously expressed in respiratory epithelia leads to reduced apical anion secretion causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to enhanced mucus accumulation in the lung and the accompanying microbial infections that ultimately cause death in CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency that, if left untreated, result in death. In addition, the majority of males with cystic fibrosis are infertile, and fertility is decreased among females with cystic fibrosis. In contrast to the severe effects of two copies of the CF associated gene, individuals with a single copy of the CF associated gene exhibit increased resistance to cholera and to dehydration resulting from diarrhea—perhaps explaining the relatively high frequency of the CF gene within the population.
Sequence analysis of the CFTR gene of CF chromosomes has revealed a variety of disease causing mutations (Cutting, G. R. et al. (1990) Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem, B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 1000 disease causing mutations in the CF gene have been identified (http://www.genet.sickkids.on.ca/cftr/app). The most prevalent mutation is a deletion of phenylalanine at position 508 of the CFTR amino acid sequence, and is commonly referred to as ΔF508-CFTR. This mutation occurs in approximately 70% of cystic fibrosis cases and is associated with a severe disease.
The deletion of residue 508 in ΔF508-CFTR prevents the nascent protein from folding correctly. This results in the inability of the mutant protein to exit the ER, and traffic to the plasma membrane. As a result, the number of channels present in the membrane is far less than observed in cells expressing wild-type CFTR. In addition to impaired trafficking, the mutation results in defective channel gating. Together, the reduced number of channels in the membrane and the defective gating lead to reduced anion transport across epithelia leading to defective ion and fluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studies have shown, however, that the reduced numbers of ΔF508-CFTR in the membrane are functional, albeit less than wild-type CFTR. (Dalemans et al. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to ΔF508-CFTR, other disease causing mutations in CFTR that result in defective trafficking, synthesis, and/or channel gating could be up- or down-regulated to alter anion secretion and modify disease progression and/or severity.
Although CFTR transports a variety of molecules in addition to anions, it is clear that this role (the transport of anions) represents one element in an important mechanism of transporting ions and water across the epithelium. The other elements include the epithelial Na+ channel (ENaC), Na+/2Cl−/K+ co-transporter, Na+—K+-ATPase pump and the basolateral membrane K+ channels that are responsible for the uptake of chloride into the cell.
These elements work together to achieve directional transport across the epithelium via their selective expression and localization within the cell. Chloride absorption takes place by the coordinated activity of ENaC and CFTR present on the apical membrane and the Na+—K+-ATPase pump and Cl− ion channels expressed on the basolateral surface of the cell. Secondary active transport of chloride from the luminal side leads to the accumulation of intracellular chloride, which can then passively leave the cell via channels, resulting in a vectorial transport. Arrangement of Na+/2Cl−/K+ co-transporter, Na+—K+-ATPase pump and the basolateral membrane K+ channels on the basolateral surface and CFTR on the luminal side coordinate the secretion of chloride via CFTR on the luminal side. Because water is probably never actively transported itself, its flow across epithelia depends on tiny transepithelial osmotic gradients generated by the bulk flow of sodium and chloride.
As discussed above, it is believed that the deletion of residue 508 in ΔF508-CFTR prevents the nascent protein from folding correctly, resulting in the inability of this mutant protein to exit the ER and traffic to the plasma membrane. As a result, insufficient amounts of the mature protein are present at the plasma membrane and chloride transport within epithelial tissues is significantly reduced. In fact, this cellular phenomenon of defective ER processing of ABC transporters by the ER machinery has been shown to be the underlying basis not only for CF disease but for a wide range of other isolated and inherited diseases.
N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide (Compound 1) is a potent and selective CF FR potentiator of wild-type and mutant (including, but not limited to, e.g., ΔF508 R117H CFTR, G551D CFTR, G178R CFTR, S549N CFTR, S549R CFTR, G551S CFTR, G970R CFTR, G1244E CFTR, S1251N CFTR, S1255P CFTR, and G1349D CFTR) forms of human CFTR. N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide is useful for treatment of patients age 6 years and older with cystic fibrosis and one of the following mutations in the CFTR gene: G551D CFTR, G1244E CFTR, G1349D CFTR, G178R CFTR, G551S CFTR, S1251N CFTR, S1255P CFTR, S549N CFTR, S549R CFTR, or R117H CFTR
Accordingly, stable bioavailable forms of Compound 1 that can be manufactured easily, including co-crystals comprising N-[2,4-bis(1,1-dimethylethyl)-5-hydroxyphenyl]-1,4-dihydro-4-oxoquinoline-3-carboxamide, and pharmaceutical compositions thereof, may be useful for developing products and/or methods for treating patients suffering from CF thereof.
In one aspect, the disclosure provides a co-crystal comprising Compound 1 and a co-former, wherein Compound 1 is represented by the following formula:
and the co-former is chosen from the following structural formula:
                wherein R1, R2, and R3 are independently C1-29 aliphatic.        
In some embodiments, the co-crystal is isolated.
In some embodiments, in the co-crystal, the stoichiometry of Compound 1 to the co-former ranges from 2 to 1 to 6 to 1.
In some embodiments, the stoichiometry of Compound 1 to the co-former in the co-crystal is 6 to 1.
In some embodiments, the stoichiometry of Compound 1 to the co-former in the co-crystal is about 6 to about 1.
In some embodiments, the stoichiometry of Compound 1 to the co-former in the co-crystal is 3 to 1.
In some embodiments, the stoichiometry of Compound 1 to the co-former in the co-crystal is about 3 to about 1.
In some embodiments, Compound 1 may form hexameric supermolecules (hexamers) in the co-crystal, wherein each of the hexamers contains six molecules of Compound 1 bound by hydrogen bonds as shown in FIG. 1.
In some embodiments, the co-crystals are capable of yielding a concentration of Compound 1 of greater than 0.4 mg/mL when dissolved in simulated intestinal fluid in fed state (FeSSIF).
In some embodiments, the co-crystals are capable of yielding a concentration of Compound 1 of greater than 0.4 mg/mL when dissolved in simulated intestinal fluid in fed state (FeSSIF) and the concentration is maintained for at least 10 hours.
In some embodiments, the co-crystals are characterized as having an X-ray powder diffraction pattern with characteristic peaks expressed in 2-theta±0.2 degrees at the following positions: 3.5, 6.9, and 10.9.
In yet some embodiments, the co-crystals are characterized as having a 13C ssNMR spectrum with characteristic peaks expressed in ppm±0.1 at the following positions: 178.6, 155.0, and 119.4.
In yet some other embodiments, the co-crystals are characterized as having a 13C ssNMR spectrum with characteristic peaks expressed in ppm±0.1 at the following positions: 178.6, 155.0, 130.5, and 119.4.
Another aspect of the present disclosure provides for pharmaceutical compositions comprising a therapeutic effective amount of Compound 1 and a pharmaceutically acceptable carrier or excipient, wherein at least 30% of Compound 1 is present in the form of co-crystals disclosed herein.
In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent.
For example, in one embodiment, the additional therapeutic agent is selected from a mucolytic agent, a bronchodilator, an antibiotic, an anti-infective agent, an anti-inflammatory agent, a CFTR modulator other than Compound 1, or a nutritional agent, or combinations thereof. In another embodiment, the additional therapeutic agent is a CFTR modulator other than Compound 1.
Further as an example, in one embodiment, the CFTR modulator is (3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid or (R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.
In another aspect, the present disclosure provides for a method treating or lessening the severity of a disease in a patient, wherein said disease is selected from cystic fibrosis, hereditary emphysema, COPD, or dry-eye disease, the method comprising the step of administering to the patient an effective amount of any of the co-crystals presented herein. For example, in one embodiment, the disease is cystic fibrosis.
In some embodiments, the method further comprises co-administering one or more additional therapeutic agents to the subject. For example, in one embodiment, the additional therapeutic agent is (3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid or (R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide. In another embodiment, the additional therapeutic agent is administered concurrently with, prior to, or subsequent to the co-crystal.
Another aspect of the present disclosure provides for a method of preparing a co-crystal comprising Compound 1 and a co-former, wherein Compound 1 is represented by the following structural formula:
the co-former is chosen from the following structural formula:

wherein R1, R2, and R3 are independently C1-29 aliphatic
comprising the step of:
combining Compound 1 and the co-former to form the co-crystal.
One aspect of the present disclosure provides for a method of preparing a co-crystal comprising Compound 1 and a co-former, wherein Compound 1 is represented by the following structural formula:
the co-former is chosen from the following structural formula:

wherein R1, R2, and R3 are independently C1-29 aliphatic.
Another aspect of the present disclosure provides for a method of preparing co-crystals comprising Compound 1 and a co-former, wherein Compound 1 is represented by the following structural formula:
the co-former is chosen from the following structural formula:

wherein R1, R2, and R3 are independently C1-29 aliphatic.
comprising the steps of:
                (a) preparing a mixture comprising Compound 1 and the co-former; and        (b) heating the mixture to 80° C.        
Further another aspect of the present disclosure provides for a method of preparing co-crystals comprising Compound 1 and a co-former, wherein Compound 1 is represented by the following structural formula:
the co-former is chosen from the following structural formula:

wherein R1, R2, and R3 are independently C1-29 aliphatic,
comprising the steps of:
                (a) preparing a mixture comprising Compound 1 and the co-former; and        (b) heating the mixture to a temperature that is about 5 to 10° C. higher than the melting point of the co-former.        
One aspect of the present disclosure provides for a method of preparing co-crystals comprising Compound 1 and a co-former, wherein Compound 1 is represented by the following structural formula:
the co-former is chosen from the following structural formula:

wherein R1, R2, and R3 are independently C1-29 aliphatic;
comprising the steps of:
                (a) preparing a mixture comprising Compound 1 and the co-former;        (b) heating the mixture;        (c) cooling down the mixture; and        (d) repeating step (b) and (c).        
Another aspect of the present disclosure provides for a method of preparing a co-crystal comprising Compound 1 and a co-former, wherein Compound 1 is represented by the following structural formula:
the co-former is chosen from the following structural formula:

wherein R1, R2, and R3 are independently C1-29 aliphatic,
comprising the steps of:
                (a) preparing a mixture comprising Compound 1 and the co-former;        (b) heating the mixture to 80° C.;        (c) cooling the mixture down to 40° C.; and        (d) repeating step (b) and (c).        
In some embodiments, the mixture comprising Compound 1 and the co-former is heated for 12 hours. In other embodiments, the mixture comprising Compound 1 and the co-former is heated for at least 12 hours. In some embodiments, the mixture comprising Compound 1 and the co-former is heated for 24 hours. In other embodiments, the mixture comprising Compound 1 and the co-former is heated for at least 24 hours.
In some embodiments, co-crystals disclosed herein, such as Compound 1:triglyceride co-crystals, may exhibit several advantages. For example, Compound 1:triglyceride co-crystals may show a better maintenance of the supersaturation than both the neat amorphous and solid amorphous dispersed form of Compound 1 (Compound 1 SDD) over longer time periods. Further as an example, in-vivo the Compound 1:triglyceride co-crystals may be metabolized in the small intestine by lipid esterase (lipases), which would effectively remove the triglycerides and further boost the Compound 1 concentration according to Le-Chatelier's principle.
In some embodiments, co-crystals disclosed herein, such as Compound 1:triglyceride co-crystals, may have the following advantages over the solid amorphous dispersed form (Compound 1 SDD) of Compound 1: (1) the co-crystals may be formulated, stored, and used under conditions where they are thermodynamically stable; (2) a controlled crystallization may be developed that can reduces potential impurity levels (impurities include, but are not limited to, solvent); (3) a manufacturing process may be developed that is more efficient and cost effective (for example, less solvent can be used in manufacturing and a lower cost process than spray drying can be developed); and (4) a stabilizing polymer may not be required for formulating co-crystals.